This invention relates to a process for producing an immunoglobulin enriched whey protein fraction from whey protein solutions. More particularly, it relates to the production of a whey protein fraction enriched in immunoglobulins, and optionally a whey protein isolate, using a cation exchanger under selected conditions.
Methods for isolating and purifying immunoglobulins from source materials are well known in the art. For example, physical methods of separating immunoglobulins from serum are known. Separation may be effected based on physical properties such as molecular weight, isoelectric point, electrophoretic mobility and solubility in various systems.
It is recognised that milk products contain, among other things, a mixture of different proteins including immunoglobulin. A process for producing immunoglobulin enriched milk products, and particularly the whey left from cheese and casein making, is commercially desirable. Many methods are known whereby whey proteins can be recovered from whey by ion exchange either as mixtures of proteins or a particular protein selected in preference to others.
Whether or not particular protein binds to an ion exchanger depends on a large number of parameters including the isoelectric point (IEP) of that protein, the pH of the protein solution and especially the choice of ion exchanger. A protein which binds to a cation exchanger at a particular pH will not usually bind to an anion exchanger at the same pH. These effects are well known to those skilled in the art and are summarised in Table 1 for the major whey proteins. It will be appreciated that this is an approximate guide to the behaviour of whey proteins on ion exchangers as there are also other factors which influence this behaviour. Furthermore, the information in the art has largely been determined empirically.
Immunoglobulin (Ig), xcex1-Lactalbumin (xcex1-LA), xcex2-Lactoglobulin (xcex2-LG), Bovine serum albumin (BSA), Glycomacropeptide (GMP present only in sweet wheys).
Given the isoelectric point of immunoglobulins of 6 to 8, their expected behaviour is that they will bind to cation exchangers at pH 3 to 7, but will not bind to anion exchangers. Indeed, immunoglobulins are the most difficult of whey proteins to bind to an anion exchanger.
In keeping with this teaching, EP Patent No. 0320 152 discloses a process for producing a whey protein concentrate enriched in immunoglobulins by contacting an anion exchanger with whey or whey protein concentrate at pH 5.5 to 7.0 preferably pH 6.0 to 6.4. After contact with the exchanger, the unadsorbed protein fraction (effluent) contained a higher proportion of immunoglobulins than the original whey or concentrate as a result of the preferential adsorption of the other major whey proteins.
It should also be noted that when sweet whey is used in the anion exchange process of EP 0 320 152 glycomacropeptide (GMP) can be adsorbed along with the major whey proteins (as long as sufficient capacity is available), so the presence of GMP does not significantly affect the level of immunoglobulin enrichment.
It would be useful to have available an alternate process for producing an immunoglobulin enriched effluent using a cation exchanger.
Also consistent with this teaching are Japanese Patent No. 2104533 and British Patent No. 2179947 which describe processes wherein whey protein solutions are contacted with cation exchangers in the pH range 5-8 such that immunoglobulin is preferentially extracted by, and recovered in high purity from the ion exchanger. The utility of cation exchangers to adsorb immunoglobulins is further identified in GB 1,563,990 and FR 2,452,881. These patents reflect the teachings in the art that cation exchangers are used to adsorb immunoglobulins.
In other published work with cation exchangers, where contact with whey is made at lower pH e.g. 3-4.5, it has been reported that the protein mixture called whey protein isolate (WPI), recovered from the cation exchanger contains the major whey proteins xcex2-lactoglobulin, xcex1-lactalbumin, BSA and immunoglobulins in the same ratio as in the whey from which it was made. (Howell et al. Dairy Products Technical Conference, 1990, pages 117-128, Wisconsin Centre for Dairy Research, Madison). Clearly immunoglobulins are usually adsorbed by the cation exchanger used to manufacture WPI.
All this is in keeping with the expected behaviour of whey proteins with ion exchangers as set out in Table 1. In particular immunoglobulins do not bind easily to anion exchangers and so can be recovered at elevated levels from the effluent (breakthrough fraction). With cation exchangers they are usually adsorbed and recovered from the ion exchanger with or without other proteins depending on the conditions used.
An exception to this is a report by A D A Kanekanian and M J Lewis (in Developments in Food Proteins, (B J F Hudson, Ed.) vol. 4, pages 135-173, Elsivier, London 1986) that CM Cellulose adsorbed all the xcex2-lactoglobulin, xcex1-lactalbumin and BSA from demineralised whey, at pH 3, but not the immunoglobulin fraction. However, the conditions identified either are not specified in detail although they do indicate an under loading of the cation exchanger with adsorbable protein. Further, the reference teaches that ion-exchange processes are mainly useful for solutions containing low concentrations of proteins (less than 1%) and that column ion-exchange systems using both an anion and cation exchanger are preferred for use, the anion exchanger to bind most of the whey proteins and the cation exchanger to bind the immunoglobulin.
Further, U.S. Pat. No. 4,834,994 discloses a method of removing xcex2-lactoglobulin selectively from whey by adsorption onto CM Cellulose under very specific conditions of pH 4.3-4.6, 60-90% demineralisation and a protein concentration of 0.5 to 1.5%. The authors point out that the whey breakthrough stream has an increased ratio of xcex1-lactalbumin to xcex2-lactoglobulin, because of the preferential adsorption and removal of xcex2-lactoglobulin. The recovered xcex2-lactoglobulin was reported as containing almost no immunoglobulin. The authors report that CM cellulose is not porous enough to bind immunoglobulin. However the applicants have shown otherwise in Example 9.
Neither this patent nor the Lewis reference make any mention of the possibility of producing a breakthrough fraction with enriched levels of imununoglobulins. Furthermore, their methods may be limited to their conditions of use.
NZ Patent No. 241328 also discloses a method of removing xcex2-lactoglobulin from whey and more particularly producing an xcex1-lactalbumin enriched cation exchanger-passed solution (breakthrough) at a non-specified temperature and under specific conditions which include a lengthy contact time of 20 hours (see Example 1).
No mention is made of whether the immunoglobulin finishes up with the xcex2-lactoglobulin or in the xcex1-lactalbumin enriched stream. The latter would make it enriched also in immunoglobulin. It cannot be assumed that if xcex1-lactalbumin does not neither will immunoglobulin. In fact, the opposite might be assumed on the basis of Table 1, that the immunoglobulin would bind with the xcex2-lactoglobulin. The applicants have shown (comparative Example 2) that under the conditions disclosed to produce an xcex1-lactalbumim enriched stream from whey, the immunoglobulin is spread into both products and that the level of enrichment and yield is not useful.
What the applicants have now surprisingly found is that for cation exchangers, which normally adsorb immunoglobulin from whey, it is possible to reduce or prevent this adsorption taking place by contacting them with an excess of adsorbable protein. Under such conditions they have found that the immunoglobulin has difficulty binding and can be recovered as a breakthrough solution whose protein content is enriched in immunoglobulin.
This is particularly so when the whey protein solution used has a higher protein concentration than that found in whey such as an ultrafiltration retentate or reconstituted WPC. The use of such concentrated solutions has the added advantage of making it easier to contact an excess of adsorbable protein with the cation exchanger and to allow shorter contact times.
The net result of this is a process with a high yield of immunoglobulin in the WPC and a significant level of enrichment.
It is therefore an object of the present invention to provide a process for producing an immunoglobulin enriched fraction from whey protein solutions which goes some way towards achieving these desiderata, or to at least provide the public with a useful choice.
In a first aspect, the present invention provides a process for producing an inmunoglobulin enriched whey protein fraction and optionally a whey protein isolate (WPI), which process comprises:
(a) subjecting a whey protein solution to ion exchange using a cation exchanger at pH 2.5-4.5 under conditions which overload the cation exchanger with potentially adsorbable protein and which cause the exchanger to adsorb preferentially whey proteins other than immunoglobulin,
(b) recovering the breakthrough whey protein fraction not adsorbed by said ion exchange step (a); and optionally
(c) eluting and recovering WPI adsorbed to said cation exchanger.
In a further embodiment, the present invention may be broadly said to consist in an immunoglobulin enriched whey protein fraction whenever prepared by a process of the invention.
For the purposes of this specification the expression xe2x80x9cto overloadxe2x80x9d means to load an ion exchanger with a quantity of potentially adsorbable protein in excess of that which the ion exchanger is able to adsorb. This results in less than the maximum yield of adsorbable protein.